RESEARCH Open Access

Heat shock protein 70 increases cellproliferation, neuroblast differentiation, andthe phosphorylation of CREB in thehippocampusHyun Jung Kwon1†, Woosuk Kim2†, Hyo Young Jung3, Min Soo Kang4, Jong Whi Kim3, Kyu Ri Hahn3,Dae Young Yoo5, Yeo Sung Yoon3, In Koo Hwang3* and Dae Won Kim1*

Abstract

In the present study, we investigated the effects of heat shock protein 70 (HSP70) on novel object recognition, cellproliferation, and neuroblast differentiation in the hippocampus. To facilitate penetration into the blood–brainbarrier and neuronal plasma membrane, we created a Tat-HSP70 fusion protein. Eight-week-old mice receivedintraperitoneal injections of vehicle (10% glycerol), control-HSP70, or Tat-HSP70 protein once a day for 21 days. Toelucidate the delivery efficiency of HSP70 into the hippocampus, western blot analysis for polyhistidine wasconducted. Polyhistidine protein levels were significantly increased in control-HSP70- and Tat-HSP70-treated groupscompared to the control or vehicle-treated group. However, polyhistidine protein levels were significantly higher inthe Tat-HSP70-treated group compared to that in the control-HSP70-treated group. In addition, immunohistochemicalstudy for HSP70 showed direct evidences for induction of HSP70 immunoreactivity in the control-HSP70- and Tat-HSP70-treated groups. Administration of Tat-HSP70 increased the novel object recognition memory compared to untreatedmice or mice treated with the vehicle. In addition, the administration of Tat-HSP70 significantly increased the populationsof proliferating cells and differentiated neuroblasts in the dentate gyrus compared to those in the control or vehicle-treated group based on the Ki67 and doublecortin (DCX) immunostaining. Furthermore, the phosphorylation of cAMPresponse element-binding protein (pCREB) was significantly enhanced in the dentate gyrus of the Tat-HSP70-treatedgroup compared to that in the control or vehicle-treated group. Western blot study also demonstrated the increases ofDCX and pCREB protein levels in the Tat-HSP70-treated group compared to that in the control or vehicle-treated group.In contrast, administration of control-HSP70 moderately increased the novel object recognition memory, cell proliferation,and neuroblast differentiation in the dentate gyrus compared to that in the control or vehicle-treated group. These resultssuggest that Tat-HSP70 promoted hippocampal functions by increasing the pCREB in the hippocampus.

Keywords: cAMP response element-binding protein, Cell proliferation, Heat shock protein 70, Hippocampus, Neuroblastdifferentiation, Novel object recognition

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: vetmed2@snu.ac.kr; kimdw@gwnu.ac.kr†Hyun Jung Kwon and Woosuk Kim contributed equally to this work.3Department of Anatomy and Cell Biology, College of Veterinary Medicine,and Research Institute for Veterinary Science, Seoul National University, Seoul08826, South Korea1Department of Biochemistry and Molecular Biology, Research Institute ofOral Sciences, College of Dentistry, Gangneung-Wonju National University,Gangneung 25457, South KoreaFull list of author information is available at the end of the article

Laboratory Animal ResearchKwon et al. Laboratory Animal Research (2019) 35:21 https://doi.org/10.1186/s42826-019-0020-2

IntroductionNeural stem cells (NSCs) are located in the subventricu-lar zone of the lateral ventricle and in the subgranularzone of the dentate gyrus, and differentiate into newneurons in the olfactory bulb and dentate gyrus [1–3].These cells are maintained throughout life, and neurogen-esis in the dentate gyrus is very important because thehippocampus is the most susceptible region to neurologicaldisorders including ischemia and Alzheimer’s diseases [4–7]. In the dentate gyrus, NSCs are located in the subgranu-lar zone, differentiate into neuroblasts, and then migrateinto the granule cell layer to become mature neurons [2].Newly generated neurons help the hippocampus to adaptfrom new environmental needs and to respond to cognitivedemands, which improves the acquisition of new skills,movement coordination, and emotional control [8, 9]. Thenumber of NSCs decreased with age, stress, and chemicaltoxins, but can be increased by exercise, environmental en-richment, and neuropsychiatric drugs such as antidepres-sants [10–14]. The modification of NSC populations iscritical for neural repair, and NSCs are believed to be a po-tential therapeutic approach for neurological disorders.Expression of heat shock protein (HSP) is induced by

abnormal conditions in various cell structures, and HSPswere named dependent on their molecular weight [15].HSP70 is a molecular chaperone that is found in the cyto-sol, nucleus, endoplasmic reticulum, and mitochondria.HSP70 has diverse functions such as protein folding, pro-tein translocation across membranes, and non-specificcytoprotection against various events [16–18]. In the ner-vous system, HSP70 and its modulators show neuropro-tective effects against neurological disorders includingAlzheimer’s disease, Parkinson’s disease, Huntington’sdisease, and stroke [19–21]. Chronic administration of ex-ogenous HSP70 improves learning and memory in oldmice, and increases synaptophysin immunoreactivity inthe brain compared to the control group [22]. HSP70 hasa cAMP 26 responsive element (CRE) motif in its pro-moter region that can be activated by CRE binding protein(CREB) [23, 24], which is one of the most important path-ways for long-term memory formation [25, 26]. However,there has been a lack of evidence on the effects of HSP70on neurogenesis in the hippocampus.The blood–brain barrier and plasma membrane are

mostly impermeable to bioactive molecules such as pep-tides, proteins, and oligonucleotides because of theirunique structures to protect the brain and cells fromtoxicants. Conversely, therapeutics with targets locatedwithin the blood-brain barrier and plasma membraneare usually ineffective [27]. The Tat peptide from the hu-man immunodeficiency virus-1 (HIV-1) has a short, cat-ionic, and arginine-rich sequence responsible for itstranslocation property [28]. Recently, HIV-1 and its Tatprotein is thought to be one of main factors to impair

cognitive function and adult hippocampal neurogenesis.Tat inhibits hippocampal neurogenesis in the mousebrain and significantly reduces the proliferation, migra-tion, and differentiation of cultured neural precursorcells [29–31]. However, in a previous study, we demon-strated that a Tat-fusion protein was efficiently trans-duced into cells, and demonstrated functional activityacross the blood–brain barrier in the spinal cord [32]and brain [33]. In addition, Tat-HSP70 showed neuro-protective effects against ischemic damage in mice [34]and inhibited inflammatory response induced by ische-mia [35]. In the present study, we created a Tat-HSP70fusion protein and observed the effects of Tat-HSP70 onnovel object recognition memory as well as on cell pro-liferation and neuroblast differentiation in the hippo-campal dentate gyrus.

Material and methodExperimental animalsForty male C57BL/6 J mice (7 weeks of age) were pur-chased from Jackson Laboratory Co. Ltd. (Bar Harbor,ME, USA). Five animals were housed per cage in a con-ventional area under standard conditions at ambienttemperature (22 ± 2 °C) and humidity (60 ± 5%) on a 12:12 h light/dark cycle with ad libitum access to food andwater. Animal handling and care conformed to guide-lines of current international laws and policies (NationalInstitutes of Health Guide for the Care and Use of La-boratory Animals, Publication No. 85–23, 1985, revised1996) and were approved by the Institutional AnimalCare and Use Committee of Seoul National University(SNU-170807-10). All experiments were conducted withan effort to minimize the number of animals used and thephysiological stress caused by the procedures employed.All experimental procedures were conducted according toARRIVE guidelines [36].

Construction of expression vectorsA cell-permeable Tat expression vector was prepared inthe present laboratory as previously described [37]. ThecDNA sequence for human HSP70 was amplified bypolymerase chain reaction (PCR) using the following pri-mer sequences: forward, 5′-CTC GAG ATG GCC AAAGCC-3′ and reverse, 5′-GGA TCC CTA ATC TACCTC CTC-3′. PCR products were excised, eluted (ExpinGel; GeneAll Biotechnology Co., Ltd., Seoul, Korea), andligated into a TA cloning vector (pGEM®-T easy vector;Promega Corporation, Madison, WI, USA) according tothe manufacturer’s protocol. The purified TA vectorcontaining human HSP70 cDNA was ligated into theTat expression vector to produce a Tat-HSP70 fusionprotein. In a similar fashion, a control HSP70 was con-structed that expressed the HSP70 protein without Tat.To produce the Tat-HSP70 and control HSP70 proteins,

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the plasmid was transformed into Escherichia coli BL21cells. The transformed bacterial cells were grown in 100mL of lysogeny broth media at 37 °C to a D600 value of0.5–1.0, and then induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 37 °C for 6 h. Harvested cellswere lysed by sonication and purified using a Ni2+-nitri-lotriacetic acid Sepharose affinity column (Qiagen, Inc.)and PD-10 column chromatography (GE Healthcare,Chicago, IL, USA). The purified protein concentrationswere estimated using a Bradford assay [38].Equal amounts of proteins were analyzed using 10%

sodium dodecyl sulfate polyacrylamide gel electrophoresis.Analyzed proteins were electrotransferred to a polyvinyli-dene difluoride membrane, and then the membrane wasblocked with tri-buffered saline (25mM Tris-HCl, 140mMNaCl, 0.1% Tween 20, pH 7.5) containing 5% non-fat drymilk. The membrane was probed using polyhistidine anti-bodies (1:2000, His-probe, SantaCruz Biotechnology, SantaCruz, CA, USA). Proteins were identified using chemilu-minescent reagents as recommended by the manufacturer(Amersham, Franklin Lakes, NJ, USA).

Administration of tat-HSP70The mice were divided into four groups: control, vehicle(10% glycerol)-treated, control-HSP70-treated, and Tat-HSP70-treated group. Vehicle, 5 nmol control-HSP70, or5 nmol Tat-HSP70 was intraperitoneally administered tomice at 8 weeks of age, once a day for 3 weeks. This dosagewas adapted because Tat-HSP70 in this dosage showedneuroprotective effects against Parkinson’s disease inducedby 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatmentin mice [39].

Novel object recognition testThe testing apparatus consisted of an open box (25 cm ×25 cm × 25 cm) made of black acryl as described in theprevious study [40]. The floor was covered with wood-chip bedding, which was moved around between trialsand testing days to prevent the build-up of odor in cer-tain places. The objects to be discriminated were madeof solid metal and could not be displaced by the micedue to their weight. The objects were cleaned withbleach to remove residual odors.On the 20th day of treatment with vehicle, control-

HSP70, or Tat-HSP70, at 1 h after treatment, mice fromeach group (n = 10 per group) were allowed to explorethe apparatus for 2 min. On the testing day (21st day oftreatment), two 2-min trials were performed 1 h follow-ing the last treatment. In the “sample” trial (T1), twoidentical objects were placed in two opposite corners ofthe apparatus. A mouse was placed in the apparatus andleft to explore these two identical objects. After T1, themouse was placed back in its home cage for an inter-trial interval of 1 h. Subsequently, a “choice” trial (T2)

was performed. In T2, a new object replaced one of theobjects that was present in T1. The mice were exposedagain to two different (familiar and new) objects. Explor-ation was defined as directing the nose toward the objectat a distance of no more than 2 cm and/or touching theobject with the nose. From this measure, a series of vari-ables were then calculated: the total time spent in ex-ploring the two identical objects in T1, and the timespent in exploring two different objects in T2.The distinction between familiar and new objects in

T2 was determined by comparing the time spent explor-ing familiar object with that spent exploring new object.The discrimination index represents the difference in ex-ploration time expressed as a proportion of the totaltime spent exploring the two objects in T2.

Western blotFollowing the novel object recognition test, animals inthe control, vehicle-treated, control-HSP70-treated, andTat-HSP70-treated groups (n = 5 in each group) weresacrificed and analyzed by western blotting as describedpreviously [32, 40]. Following deep anesthesia with 2 g/kgurethane (Sigma-Aldrich, St. Louis, MO, USA) and subse-quent decapitation, the hippocampal tissues were cut into500-μm-thick sections on a vibratome (Leica MicrosystemsGmbH), and the hippocampus was cut out using a surgicalblade. The hippocampal tissues were homogenized in50 mM phosphate-buffered saline (PBS, pH 7.4) con-taining 0.1mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (pH 8.0), 0.2% Nonidet P-40,10mM ethylenediaminetetraacetic acid (pH 8.0), 15mMsodium pyrophosphate, 100mM β-glycerophosphate, 50mM sodium fluoride, 150mM sodium chloride, 2 mM so-dium orthovanadate, 1 mM phenylmethylsulfonyl fluoride,and 1mM dithiothreitol (DTT). Following centrifugationfor 5min at 16,000×g at 4 °C, the protein concentrationwas determined in the supernatants using a Micro BCAprotein assay kit with bovine serum albumin as the stand-ard (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA,USA). Aliquots containing 20 μg of total protein wereboiled in loading buffer containing 150mM Tris (pH 6.8),3 mM DTT, 6% sodium dodecyl sulfate, 0.3% bromophe-nol blue, and 30% glycerol. Each aliquot was subsequentlyloaded onto a polyacrylamide gel. Following electrophor-esis, the proteins in the gel were transferred to a nitrocel-lulose membrane (Pall Life Sciences, Port Washington,NY, USA). To reduce background staining, the membranewas incubated with 5% non-fat dry milk in PBS containing0.1% Tween-20 for 45min at 25 °C, which was followed byincubation with rabbit anti-polyhistidine primary antibody(1:2000, His-probe, SantaCruz Biotechnology), rabbitanti-doublecortin (DCX) antibody (1:10,000; Abcam,Cambridge, UK), rabbit anti-phosphorylated CREB atSer133 (pCREB; 1:1000; Cell Signaling Technology,

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Inc., Beverly, MA, USA), or rabbit anti-CREB (1:1000; CellSignaling Technology, Inc.), peroxidase-conjugated goatanti-rabbit IgG (1:5000, SantaCruz Biotechnology), and anECL chemiluminescent kit (Pierce; Thermo Fisher Scien-tific, Inc.).

Tissue processingFollowing the novel object recognition test, animals (n =5 in each group) were deeply anesthetized with 1 g/kg ofurethane (Sigma-Aldrich) and perfused transcardiallywith 0.1M PBS (pH 7.4) followed by 4% paraformalde-hyde in 0.1 M PBS (pH 7.4) as described previously [32,40]. The brains were dissected and post-fixed for 12 h inthe same fixative. The tissue was cryoprotected by over-night saturation with 30% sucrose. Serial brain sectionswere cut coronally at a thickness of 30 μm using a cryo-stat (Leica, Wetzlar, Germany), and collected in 6-wellplates containing PBS for further processing.

ImmunohistochemistryAll sections were processed under the same conditionsto ensure that the immunohistochemical data were com-parable among the groups. Tissue sections located at adistance of 90 μm from each other were selected froman area between 1.82 and 2.30 mm posterior to thebregma, as defined by a mouse atlas [41] for Ki67, DCX,and pCREB immunohistochemistry. The sections weresequentially treated with 0.3% H2O2 in PBS for 30 minand 10% normal goat serum in 0.05M PBS for 30 min at25 °C. Sections first underwent an overnight incubationwith mouse anti-HSP70 antibody (1:500; Calbiochem, EMDMillipore, Temecula, CA, USA), rabbit anti-Ki67 antibody(1:1000; Abcam), rabbit anti-DCX (1:5000; Abcam), orrabbit anti-pCREB (1:400; Cell Signaling Technology, Inc.)at 25 °C. Thereafter, the sections were treated with biotinyl-ated goat anti-mouse or anti-rabbit IgG and a streptavidin-peroxidase complex (1:200; Vector, Burlingame, CA, USA)for 2 h at 25 °C. Sections were visualized by reaction with 3,3′-diaminobenzidine tetrachloride (Sigma) in 0.1M Tris-HCl buffer (pH 7.2) and mounted on gelatin-coated slides.Sections were dehydrated and mounted with Canada bal-sam (Kanto Chemical, Tokyo, Japan).

Data analysisAnalysis of the hippocampal dentate gyrus for DCX wasperformed using an image analysis system and ImageJsoftware v. 1.50 (National Institutes of Health, Bethesda,MD, USA). Data analysis was carried out under the sameconditions by two observers for each experiment toensure objectivity in blinded conditions as described inthe previous study [40]. Tissue sections located at a dis-tance of 90 μm from each other were selected from anarea between 1.82 and 2.30 mm posterior to the bregma,as defined by a mouse atlas [41]. Digital images of the

whole dentate gyrus were captured with a BX51 lightmicroscope (Olympus, Tokyo, Japan) equipped with adigital camera (DP72, Olympus) connected to a computermonitor. Images were calibrated into an array of 512 ×512 pixels corresponding to a tissue area of 1200 μm×900 μm (100× primary magnification). Each pixel reso-lution had 256 Gy levels, and the intensity of DCX immu-noreactivity was evaluated by relative optical density(ROD), which was obtained after transformation of themean gray level using the following formula: ROD= log(256/mean gray level). The ROD of background stainingwas determined in unlabeled portions of the sectionsusing Photoshop CC 2018 software (Adobe Systems Inc.,San Jose, CA, USA), and this value was subtracted to cor-rect for nonspecific staining using ImageJ v. 1.50 software(National Institutes of Health). Data are expressed as apercentage of the control group (which was set at 100%).The Ki67- and pCREB-immunoreactive nuclei in the

whole dentate gyrus was counted using an analysis systemequipped with a computer-based CCD camera (OPTIMASsoftware version 6.5; CyberMetrics® Corporation, Phoenix,AZ, USA; magnification, 100×) as described in a previousstudy [42]. The image was converted to a gray-scale image,and Ki67- and pCREB-immunoreactive nuclei were auto-matically selected according to the intensity of the immu-nohistochemical staining for Ki67 and pCREB, respectively.

Statistical analysisThe data were expressed as the mean of the experimentsperformed for each experimental investigation. In orderto determine the changes of cell number and ROD, meandifferences among the groups were analyzed statistically byone-way analyses of variance followed by Bonferroni’spost-hoc test using GraphPad Prism 5.01 software (Graph-Pad Software, Inc., La Jolla, CA, USA). The results wereconsidered to be statistically significant if p < 0.05.

ResultsConstruction of control-HSP and tat-HSP70 fusion proteinsTo produce the control-HSP70 and Tat-HSP70 fusion pro-teins, human HSP70 genes were fused to a Tat peptide ex-pression vector, and the control-HSP70 protein wasmanufactured without a Tat domain (Fig. 1a). After theoverexpression of vectors, purified control-HSP70 and Tat-HSP70 fusion proteins were obtained by Nib+-→Ni2+-nitrilotriacetic acid Sepharose affinity column and PD-10column chromatography. Western blot analysis revealedstrong bands for control-HSP and Tat-HSP70 (Fig. 1b).

Confirmation of tat-HSP70 delivery into the hippocampusThe delivery of Tat-HSP70 was assessed by western blotanalysis for polyhistidine and by immunohistochemistryfor HSP70. Polyhistidine protein levels were significantlyincreased in the hippocampal homogenates of control-

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Fig. 1 Purification and transduction of the Tat-HSP70 protein into the mouse hippocampus. a Overview of the Tat-HSP70 protein. b Expressionand purification of the Tat-HSP70 and control-HSP70 proteins, as assessed by western blot analyses using an anti-polyhistidine antibody. cWestern blot analysis of polyhistidine in the hippocampal homogenates of control, vehicle (10% glycerol)-, control-HSP70-, and Tat-HSP70-treatedgroups (n = 5 per group; aP < 0.05, versus control group; bP < 0.05, versus vehicle-treated group; cP < 0.05, versus control-HSP70 treated group). dImmunohistochemistry for HSP70 in the dentate gyrus of control, vehicle-, control-HSP70-, and Tat-HSP70-treated mice. Note that HSP70immunoreactive structures are most abundantly found in the Tat-HSP70-treated group. GCL, granule cell layer; ML, molecular layer; PL,polymorphic layer. Scale bar = 50 μm

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HSP70 and Tat-HSP70-treated group compared to that incontrol or vehicle-treated group. In addition, polyhistidineprotein levels were significantly higher in the Tat-HSP70-treated group compared to that in the control-HSP70-treated group (Fig. 1c). HSP70 immunoreactivity was faintlyobserved in the dentate gyrus of control and vehicle-treatedgroup, while HSP70 immunoreactivity was abundantly ob-served in the dentate gyrus of control-HSP70 or Tat-HSP70-treated group. In addition, HSP70 immunoreactivestructures were more abundant in the dentate gyrus of Tat-HSP70-treated group compared to that in the control-HSP70-treated group (Fig. 1d).

Effects of tat-HSP70 on novel object recognition memoryDuring the training period, there were no significant dif-ferences on the amount of time spent exploring twoidentical objects among the groups. During the testingperiod, mice in all groups spent more time exploring thenew object compared with a familiar one. However, micein the control-HSP70- and Tat-HSP70-treated groups spentsignificantly more time exploring the new object comparedto that in the control or vehicle-treated groups. Thediscrimination index was also significantly higher in thecontrol-HSP70- and Tat-HSP70-treated groups comparedto that in the control or vehicle-treated groups. The dis-crimination index was significantly higher in the Tat-HSP70-treated group compared to that in the control-HSP70-treated group (Fig. 2).

Effects of tat-HSP70 on cell proliferation in the dentate gyrusIn all groups, Ki67-positive nuclei were mainly found inthe subgranular zone of dentate gyrus, but the numberof Ki67-positive nuclei was significantly different among

groups. In the control and vehicle-treated groups, someKi67 nuclei was observed in the dentate gyrus (Fig. 3aand b), and the number of Ki67-positive nuclei wasslightly increased in the vehicle-treated group comparedto that in the control group, although the statistical sig-nificance was not detected between the control andvehicle-treated group (Fig. 3e). In the control-HSP70-and Tat-HSP70-treated groups, Ki67-positive nuclei wereabundant compared to that in the control group (Fig. 3cand d). The number of Ki67-positive nuclei in the control-HSP70- and Tat-HSP70-treated groups was significantlyincreased by 181.9 and 197.1% of the control group,respectively (Fig. 3e).

Effects of tat-HSP70 on neuroblast differentiation in thedentate gyrusIn all groups, DCX immunoreactive neuroblasts werefound in the dentate gyrus. Their cell bodies were lo-cated in the subgranular zone of dentate gyrus, and theirdendrites extended into the molecular layer of dentategyrus. However, DCX immunoreactivity was significantlydifferent among the groups. In the control and vehicle-treated groups, DCX-immunoreactive neuroblasts werefound sparsely in the dentate gyrus (Fig. 4a and b), andthe DCX immunoreactivities were similar in the dentategyrus between the groups (Fig. 4e). In the control-HSP70-and Tat-HSP70-treated groups, DCX-immunoreactiveneuroblasts were abundantly found in the dentate gyrus,and DCX immunoreactivities in the control-HSP70- andTat-HSP70-treated groups were significantly increased by171.5 and 194.2% of the control group, respectively(Fig. 4c, d, and e). DCX protein levels showed significantincreases in the control-HSP70 and Tat-HSP70-treated

Fig. 2 Exploration time (n = 10 per group; *p < 0.05, significant difference between familiar and novel objects) and discrimination index (n = 10per group; aP < 0.05, versus control group; bP < 0.05, versus vehicle-treated group; cP < 0.05, versus control-HSP70 treated group) of familiar vs.novel objects during a novel object recognition test in control, vehicle-, control-HSP70-, and Tat-HSP70-treated mice. Data of the exploration timefor each object (same object, where one object was replaced by a new one on the testing day) are presented as a percentage of totalexploration time. All data are shown as % exploration time ± SEM

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group compared to that in the control group and werehighest in the Tat-HSP70-treated group (Fig. 4f).

Effects of tat-HSP70 on the phosphorylation of CREB inthe dentate gyrusIn all groups, pCREB-immunoreactive nuclei were mainlyfound in the subgranular zone of the dentate gyrus. How-ever, the number of pCREB-immunoreactive nuclei wassignificantly different among the groups. A similar patternof distribution and the number of pCREB-immunoreactivenuclei was found in the control and vehicle-treated groups(Fig. 5a, b, and e). In the control-HSP-treated group,pCREB-immunoreactive nuclei were abundant comparedto that of the control group, although the statisticalsignificance was not detected between the control andcontrol-HSP70-treated groups (Fig. 5c and e). In the Tat-HSP70-treated group, pCREB-immunoreactive nuclei were

strongly observed in the subgranular zone of the dentategyrus, and the number of pCREB-immunoreactive nucleiwas significantly increased in the dentate gyrus by 161.5and 178.3% of the control group, respectively (Fig. 5d ande). Western blot study showed the significant in-creases in pCREB/CREB ratio in the control-HSP70or Tat-HSP70-treated groups compared to that in thecontrol or vehicle-treated group. However, there wereno significant differences on the ratio of pCREB/CREB between control-HSP70 or Tat-HSP70-treatedgroups (Fig. 5f).

Discussionhsp70 can regulate expression via negative feedback [43,44], and overexpression of HSPA1A suppresses HSP70induction. Several lines of evidence demonstrate that ex-ogenous HSP70 can cross the blood–brain barrier and

Fig. 3 Immunohistochemistry for Ki67 in the dentate gyrus of control (a), vehicle- (b), control-HSP70- (c), and Tat-HSP70-treated (d) mice. In allgroups, Ki67-positive nuclei (arrows) are found in the subgranular zone of the dentate gyrus. Note that Ki67-positive nuclei are abundantlydetected in the dentate gyrus of control-HSP70 and Tat-HSP70-treated mice. GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer.Scale bar = 50 μm. e The number of Ki67-positive nuclei in the dentate gyrus per section for each group are also shown (n = 5 per group; aP <0.05, versus control group; bP < 0.05, versus vehicle-treated group). Data are presented as mean ± SEM

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protects neurons from damage due to energy deprivation[45], Alzheimer’s disease [46], and epilepsy [47]. How-ever, these studies have been conducted in pathologicalnon-physiological conditions. In the present study, wemade a Tat-HSP70 fusion protein and control-HSP70protein to deliver HSP70 into neurons, and comparedthe effects of control-HSP70 and Tat-HSP70 on novelobject recognition memory as well as cell proliferationand neuroblast differentiation in the dentate gyrus.We successfully constructed control-HSP70 and Tat-

HSP70 fusion proteins as indicated by the western blotanalysis for polyhistidine, which showed differences inexpression of polyhistidine at 1.56-kDa according to themolecular weight of the Tat peptide. To assess the deliv-ery efficiency of HSP70 into the hippocampus, we

administered control-HSP70 and Tat-HSP70 to mice for21 days and observed significant increases in the expres-sion of polyhistidine in both groups compared to that inthe control or vehicle-treated group. However, polyhisti-dine protein levels were more abundantly found in thehippocampal homogenates of the Tat-HSP70-treatedgroup compared to that in the control-HSP70-treatedgroup. In addition, we also conducted the immunohisto-chemical staining for HSP70 to elucidate direct transferof HSP70 in the hippocampus. Treatment with control-HSP70 or Tat-HSP70 showed significant increases inHSP70-immunoreactive structures compared to that inthe control or vehicle-treated group although higher pro-tein levels of HSP70 were observed in the Tat-HSP70-treated group than in the control-HSP70-treated group.

Fig. 4 Immunohistochemistry for DCX in the dentate gyrus of control (a), vehicle- (b), control-HSP70- (c), and Tat-HSP70-treated (d) mice. In allgroups, DCX-immunoreactive neuroblasts (arrows) are found in the dentate gyrus with dendrites. Note that DCX-immunoreactive neuroblastsshows delicate dendritic tree branching in the dentate gyrus of control-HSP70- and Tat-HSP70-treated mice. GCL, granule cell layer; ML, molecularlayer; PL, polymorphic layer. Scale bar = 50 μm. e The relative optical densities (RODs) expressed as a percentage of the value representing theDCX immunoreactivity in the dentate gyrus of the control group are shown. f Values from western blot analysis is expressed as a ratio of DCXand β-actin immunoblot band in control group (n = 5 per group; aP < 0.05, versus control group; bP < 0.05, versus vehicle-treated group). Data arepresented as mean ± SEM

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This result suggests that HSP70 protein can cross theblood–brain barrier in naïve mice, and that Tat-HSP70was efficiently delivered to the mouse hippocampus.In the present study, administration of control-HSP70

or Tat-HSP70 for 3 weeks significantly enhanced thenovel object recognition in the mice. This result wasconsistent with previous studies showing that exogenousHSP70 or HSP70 induces improvements in learning andmemory formation [22, 48, 49]. Intranasal administrationof exogenous HSP70 for 5 and 9months resulted in im-provements in learning and memory in old mice [22]. Inaddition, infusion of recombinant HSP70 (hspa1a) intothe dorsal hippocampus facilitates memory consolidationimmediately after training, while administration of HSP70antibody blocked the memory consolidation immediatelyafter training [49]. Geranylgeranylacetone, an inducer of

HSP70, enhanced recovery of cognitive/affective function inthe Morris water maze and novel object recognition in amouse model of traumatic brain injury [48]. HSP70 hasbeen found on the external leaflet of neuroblast membranesand regulate neuroblast migration in the subventricularzone [50]. Administration of control-HSP70 or Tat-HSP70for 3 weeks significantly increased cell proliferation andneuroblast differentiation in the dentate gyrus. However,administration of geranylgeranylacetone for 8 weeks did notshow any significant changes in DCX-immunoreactive neu-roblasts in non-stress (control) mice, although they signifi-cantly increased DCX-immunoreactive neuroblasts in thestressed mice [51]. In the ischemic animals, Tat-HSP70-transduced neural precursor cells enhanced the functionaloutcome by preventing secondary neuronal degeneration aswell as facilitating neurogenesis with sustained high levels

Fig. 5 Immunohistochemistry for pCREB in the dentate gyrus of control (a), vehicle- (b), control-HSP70- (c), and Tat-HSP70-treated (d) mice. In allgroups, pCREB-positive nuclei (arrows) are mainly observed in the subgranular zone of the dentate gyrus. Note that pCREB-positive nuclei arestrongly observed in the dentate gyrus of Tat-HSP70-treated mice. GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer. Scale bar =50 μm. e The number of pCREB-positive nuclei per section for each group are shown and f values from western blot analysis is expressed as aratio of pCREB and CREB immunoblot band in control group (n = 5 per group; aP < 0.05, versus control group; bP < 0.05, versus vehicle-treatedgroup). Data are presented as mean ± SEMs

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of growth factors including brain-derived neurotrophic fac-tor [52]. In an animal model for Alzheimer’s disease, intra-nasal administration of exogenous HSP70 increases genesinvolved in neuronal activity and neurogenesis [53].To elucidate the possible mechanisms of HSP70 for fa-

cilitating cell proliferation and neuroblast differentiationin the dentate gyrus, we have focused on the CREB path-way because activation of CREB is one of the most im-portant pathways for long-term memory formation andmemory consolidation [25, 26]. Administration of control-HSP70 or Tat-HSP70 for 3 weeks significantly increasedthe number of pCREB-immunoreactive nuclei in the sub-granular zone of dentate gyrus. This result suggests thatcontrol-HSP70 and Tat-HSP70 facilitated cell proliferationand neuroblast differentiation in the dentate gyrus by fa-cilitating the phosphorylation of CREB. CREB is an essen-tial factor for inducing the formation of synapses, long-term potentiation, and memory formation in mice [54–56]. In addition, we also observed that pCREB expressionin the subgranular zone of dentate gyrus was mainly foundin the DCX-immunoreactive neuroblasts in mice [57]. AsCREB is a downstream regulator of the extracellularsignal-regulated kinases (ERK) cascade, direct infusion ofrecombinant HSP70 into the dorsal hippocampus en-hanced ERK activity immediately after training in thehippocampus of aged mice [49].

ConclusionAdministration of Tat-HSP70 significantly improved novelobject recognition by increasing cell proliferation andneuroblast differentiation in the dentate gyrus via the en-hanced phosphorylation of CREB.

AbbreviationsCRE: cAMP 26 responsive element; CREB: CRE binding protein;DCX: Doublecortin; DTT: Dithiothreitol; ERK: Extracellular signal-regulated ki-nases; HIV-1: Human immunodeficiency virus-1; HSP: Heat shock protein;NSC: Neural stem cell; PBS: Phosphate-buffered saline; PCR: Polymerase chainreaction; pCREB: Phosphorylated CREB at Ser133; ROD: Relative opticaldensity; T1: Sample trial; T2: Choice trial

Authors’ contributionsWK, HJK, HYJ, JWK, KRH, DYY, YSY, DWK, and IKH conceived the study. WK,HJK, DWK, and IKH designed the study. WK, HYJ, JWK, and KRH conductedthe animal experiments and HJK and DWK conducted biochemicalexperiments. DYY, and YSY participated in designing and critical discussingthe study. All authors have read and approved the final manuscript.

FundingThis work was supported by the Promising-Pioneering Researcher Programthrough Seoul National University (SNU) in 2015 and by the National Re-search Foundation of Korea (NRF) grant funded by the Korea government(MSIP) (No. NRF-2016R1A2B4009156 and NRF-2018R1A2B6001941). Inaddition, this study was partially supported by the Research Institute for Vet-erinary Science of Seoul National University.

Availability of data and materialsThe datasets generated and/or analyzed during the current study areavailable from the corresponding author on reasonable request.

Ethics approval and consent to participateExperimental protocols for this study were approved by the InstitutionalAnimal Care and Use Committee of Seoul National University (SNU-170807-10).

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Biochemistry and Molecular Biology, Research Institute ofOral Sciences, College of Dentistry, Gangneung-Wonju National University,Gangneung 25457, South Korea. 2Department of Biomedical Sciences, andResearch Institute for Bioscience and Biotechnology, Hallym University,Chuncheon 24252, South Korea. 3Department of Anatomy and Cell Biology,College of Veterinary Medicine, and Research Institute for Veterinary Science,Seoul National University, Seoul 08826, South Korea. 4Department ofAnatomy, College of Veterinary Medicine, Kangwon National University,Chuncheon 24341, South Korea. 5Department of Anatomy, College ofMedicine, Soonchunhyang University, Cheonan 31151, South Korea.

Received: 20 August 2019 Accepted: 9 October 2019

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